In recent years, active research have been directed to expression of desired foreign proteins on the surface of single cell organisms, such as bacteriophages, bacteria, and yeasts, because proteins expressed on the cell surface have various useful applications, including production of novel vaccines, screening of various antigens and antibodies, immobilization of useful enzymes onto the cell surface, and the like.
At first, the expression of foreign proteins on the cell surface was applied to the screening of epitopes and antigenicity-determining peptide fragments, with the aim of stable vaccine production. Before then, the production of vaccine had been achieved by the selection of the mutants, which showed stable and continuous titers from the randomly mutated pathogen library. However, the vaccines produced in the conventional manner are likely to lose their antigenicity when being administered via oral routes to humans or animals. Much effort has been made to overcome these problems using the live oral vaccines displayed on the cell surface.
In one strategy to express antigenic proteins on the cell surface, endogenous cell surface proteins are used to guide these proteins onto the cell surface. For example, a gene encoding a cell surface protein is fused to a gene encoding an antigenic protein, and the resulting recombinant gene is introduced into Gram-negative bacteria to express the fusion protein on the cell surface. The antigenic protein passenger in the fusion protein vehicle can act as an effective antigen to elicit immune responses. Especially, Gram-negative bacteria are very efficient for this purpose, because the lipopolysaccharide (LPS) presenting on their cell envelope enhances the antigenicity of the fusion protein expressed on the cell surface.
As a rule, proteins to be secreted or expressed on cell surfaces have secretion signals, which allow the nascent proteins to pass through the cytoplasmic membrane, in their primary sequence. To be expressed on the cell surface of Gram-negative bacteria, a protein translocates across the cytoplasmic membrane and the periplasmic space and embedded in the outer membrane so as to protrude to the outer membrane. In bacteria, several enzymes and toxins have secretion signals and (or) targeting signals, which induce the proteins to target regions. Therefore, using such a secretion signal or targeting signal with the suitable promoter can successfully perform localization of a foreign protein on cell surfaces.
Thus far, extensive attempts have been made to utilize surface proteins of Gram-negative bacteria in expressing foreign proteins of interest onto the cell surface. The surface proteins used for the localization of foreign proteins can be largely classified into outer membrane proteins, lipoproteins, secretory proteins, and cell surface organelle proteins.
LamB, PhoE and OmpA, known as outer membrane proteins of Gram-negative bacteria, have been used for the production of foreign proteins on cell surfaces. When the outer membrane proteins are employed, foreign proteins are limited in size, because they must be inserted into the loops protruded out of the cell surface. Additionally, since the C- and N-termini of the foreign proteins to be inserted are required to be located near each other in the three dimensional structure, both termini must be brought close to each other by the use of a peptide linkage when the distance therebetween is large.
In fact, where LamB or PhoE is used, insertion of a foreign polypeptide as large as or larger than 50–60 amino acids failed to construct a stable membrane protein by the steric hindrance. (Charbit et al., J. Immunol., 1997, 139, 1658–1664; Agterberg et al., Vaccine, 1990, 8, 85–91). To solve this problem, an OmpA fragment containing a minimum target signal necessary for proper location was tried. For example, β-lactamase linked to the C-terminus of a target signal of OmpA was successfully expressed on the cell surface. Protein translocation from the cytoplasm to the outer membrane was achieved by the fusion of the signal sequence for the lipoprotein Lpp of E. coli to the N-terminus of OmpA (Francisco et al., Proc. Natl. Acad. Sci. USA, 1992, 489, 2713–2717).
As described above, use of bacterial outer membrane proteins in cell surface display of foreign proteins requires linkage between the foreign proteins and appropriate outer membrane proteins at a gene level, so as to synthesize fusion proteins capable of passing through the cytoplasmic membrane and being stably embedded in the outer membrane. Suitable surface anchoring motif is an outer membrane protein which satisfies the following requirements: 1) to have a secretion signal which allows the fusion protein to pass through the cytoplasmic membrane; 2) to have a target signal which allows the fusion protein to anchor in the outer membrane; 3) to be expressed on the outer membrane in large quantities; and 4) to be expressed stably irrespective of protein size. Thus far, a surface-anchoring motif, which meets all of the requirements, has not yet been developed.
Meanwhile, lipoproteins have also been used as a surface-anchoring motif. Especially, lipoprotein from E. coli is very useful, because it translocates across the inner membrane by the N-terminal secretion signals and directly linked to outer or inner membrane lipids via the covalent bond of its terminal L-cystein. Lpp, a major lipoprotein from E. coli, which is associated with the outer membrane at its N-terminus and with the cell wall peptidoglycan (PG) at its C-terminus, can be used to secrete and transport foreign proteins onto the surface of E. coli by fusion with the outer membrane protein A (OmpA, Francisco et al., Proc. Natl. Acad. Sci. USA, 1992, 489, 2713–2717). Another lipoprotein used in the surface expression of foreign protein is TraT. It was reported that TraT has been used to express peptides such as polioviral C3 epitope on the cell surface of E. coli (Felici et al., J. Mol. Biol., 1991, 222, 301–310). Additionally, a peptidoglycan-associated lipoprotein (PAL), whose function has not been elucidated clearly yet, was used for the surface expression of a recombinant antibody (Fuchs et al., Bio/Technology, 1991, 9, 1369–1372). In this case, C-terminus of the PAL was associated with peptidoglycan and N-terminus of it was fused to the recombinant antibody exposed on the cell surface.
Secretory proteins, which pass through the outer membrane, may be used as the surface anchor, but these are not well developed in Gram-negative bacteria. Only a few proteins have the secretory mechanisms by aid of the helper proteins. For instance, the lipoprotein pullulanase secreted from Klebsiella oxytoca is anchored on the outer membrane via a linkage between the lipid and its N-terminus, and completely secreted into a culture medium during the growth-resting phase. Kornacker et al. have been tried to express P-lactamase on the cell surface using the N-terminal fragment of pullulanase, but the expressed pullulanase-β-lactamase fusion protein was released to the cell media after short period of anchoring. When using alkaline phosphatase, a periplasmic space protein, as a target protein, the surface expression was not achieved. Functional expression of alkaline phosphatase appears to be difficult because at least 14 proteins are necessary for the secretion of this protein (Kornacker et al., Mol. Microl., 1990, 4, 1101–1109).
The IgA protease derived from Neisseria, a pathogenic microorganism possessing an interesting secretion system, has a secretion signal at the C-terminal beta-domain and this signal guides the N-terminal protease domain onto the cell surface. This protease is secreted into the culture medium by its own catalytic hydrolysis after being anchored on the outer membrane. Using the IgA protease beta-subunit, the 12 kDa form of cholera toxin B-subunit was surface expressed (Klauser et al., EMBO J., 1990, 9, 1991–1999). However, the protein folding occurred in the periplasmic space during the transport prevents the secretion of the fusion protein.
Proteins from cell surface organelles of Gram-negative bacteria, such as flagella, pili, and fimbriae, may be also available as surface anchoring motifs. For example, a flagellin, the subunit protein of flagellar filament, was used for the stable surface expression of cholera toxin B subunit and a B-type hepatitis viral peptide, and which were found to strongly react with their corresponding antibodies (Newton et al., Science, 1989, 244, 70–72). When using fimbrilin, a fimbria subunit protein, as a surface anchoring motif, only small size peptides were successfully expressed (Hedegaard et al., Gene, 1980, 85, 115–124).
Similar attempts have recently been developed in Gram-positive bacteria using surface proteins of Gram-positive and negative bacteria as surface anchoring motifs (Samuelson et al., J. Bacterial., 1995, 177, 1470–1476). A malaria blood stage antigen consisting of 80 amino acid residues and a albumin-associated protein from Streptococcus protein G were effectively expressed on the cell surface of Gram-positive bacteria using a surface expression system containing the Staphylococcus aureus-derived Protein A as a surface anchoring motif and a secretion signal from Staphylococcus hyicus-derived lipase.
As a result of extensive research on surface expression in Gram-positive and Gram-negative bacteria, various surface expression systems are developed and patented in the U.S.A., Europe, and Japan. In the past three years, eight patents concerning surface expression systems have been issued, among which, the case using outer membrane proteins of Gram-negative bacteria is five (WO9504069, WO9324636, WO9310214, EP603672 and U.S. Pat. No. 5,356,797), using a pilus, a cell surface organelle, is one (WO9410330), and using a cell surface lipoprotein is one (WO9504079).
The most widely used bacterial host for foreign protein production is E. coli because it is easy to culture and its gene structure is well known. Foreign proteins, however, are not well secreted into culture media under ordinary conditions of E. coli. Additionally, when foreign proteins are excessively expressed, they are accumulated as inclusion bodies within the cell. Accordingly, the purification of them requires a refolding process for solubilizing the inclusion bodies, which results in a significant reduction in yield. Further, since E. coli produces endotoxins harmful to the human body, the recombinant proteins may be contaminated with the toxins when being purified.
In contrast, yeast has been studied as a host for producing useful foreign proteins by genetic engineering techniques because it can easily secrete proteins in active forms into the culture media under the control of its own intracellular secretion system, which is operated in a manner similar to that of higher eucaryotic organisms.
Since the production of interferon in 1981 (Hitzeman et al., 1981, nature, 293; 717–722), yeast has been extensively utilized for the production of foreign proteins. In addition, not only were recombinant proteins of yeast approved by the FDA of the U.S.A. for their safety to the human body, but also most of the regulatory mechanisms of gene expression in yeast are known (Strathern et al., The Molecular Biology of the Yeast Saccharomyces, Metabolism and Gene Expression, Cold Spring Harbor Laboratory, N.Y., 1982). Accordingly, yeast system provides several significant advantages for the production of foreign proteins. For example, the proteins expressed in yeast are safe to the human body, and extracellularly secreted in conformations retaining high specific activity, similar to those expressed in animal or human cells. Furthermore, the purification processes for the proteins produced from yeast is simple compared to E. coli, and requires no refolding processes to obtain active forms, thus showing high production yield. Particularly, the surface expression in S. cerevisiae has recently been under extensive study. A few years ago, studies on the surface expression of foreign proteins in yeast were mainly focused on α-agglutinin, a typical cell wall protein as a surface anchoring motif (Schreuder et al., Yeast, 1993, 9, 399). In recent years, the study about surface anchoring motif has been extended to various cell wall proteins. Above all, the screening of surface anchor proteins through the conserved sequence analysis is extensively achieved, as the genome project for S. cerevisiae has been completed (Hamada et al., Mol. Gen. Genet., 1998, 258, 53). Using such surface proteins as surface anchoring motifs, various enzymes, including α-galactosidase, glucoamylase, lipase, and cutinase have been stably expressed on the cell surface of S. cerevisiae. In addition, expression of various enzymes on cell surface could develop many useful industrial biocatalysts (Murai et al., Appl. Microbiol. Biotechnol., 1999, 51, 65). A surface expression system using α-agglutinin as an anchoring motif has been developed and commercialized by Invitrogen Corporation. Furthermore, yeast, which is a eucaryotic microorganism usually harmless to the human body, is highly useful as a host for producing proteins for use in food or medical materials. For instance, B-type hepatitis viral antigen (HbsAg) was expressed on the yeast cell surface with the aim of developing live vaccines (Schreuder et al., Vaccine, 1996, 14, 383).
As mentioned above, active research has been directed to cell wall proteins throughout the world, but limited to S. cerevisiae. At present, researches on the cell wall proteins of other yeasts, such as Candida albicans, are in the initial stage. Therefore, there remains an urgent need for studies on surface proteins and surface expression thereof in industrially useful yeasts. Because the mediating proteins that have been studied thus far make use of glycosylphosphatidylinositol-anchor for surface anchoring, the proteins of interest must be linked to the anchor proteins at their carboxyl termini. However, some of the proteins may not exhibit their full activity under such a condition, and this is recognized as a drawback in yeast surface expression system.